Miniaturized, multiplexed readout of droplet-based microfluidic assays using time-domain modulation.

Recent advances in microfluidics to generate and control picoliter emulsions of water in oil have enabled ultra-sensitive assays for small molecules, proteins, nucleic acids, and cells. Unfortunately, the conventional fluorescence detection used to measure the outcome of these droplet-based assays has not proven suited to match the time and space multiplexing capabilities of microfluidic systems. To address this challenge, we developed an in-flow fluorescence detection platform that enables multiple streams of droplets to be monitored using only a single photodetector and no lenses. The key innovation of our technology is the amplitude modulation of the signal from fluorescent droplets using distinct micro-patterned masks for each channel. By taking advantage of the high bandwidth of electronics, our technique enables the velocity-independent recovery of weak fluorescent signals (SNR ≪ 1) using only simple hardware, obviating the need for lasers, bulky detectors, and complex fluid control. We demonstrated a handheld-sized device that simultaneously monitors four independent channels with the capability to be scaled-up to more than sixteen, limited primarily by the droplet density.